Treatment solution and method for preventing posterior capsular opacification by selectively inducing detachment and/or death of lens epithelial cells

ABSTRACT

A treatment solution used to prevent posterior capsular opacification is applied or introduced into the lens capsular bag before, during, or after cataract surgery. The treatment solution may also be applied to an intraocular lens prior to surgery. The treatment solution comprises an ion transport mechanism interference agent, which either alone or in combination with other treatment agents such as an osmotic stress agent and an agent to establish a suitable pH, selectively induces detachment and/or death of lens epithelial cells such that posterior capsular opacification is prevented. While the ion transport mechanism interference agent is capable of interfering with the cellular mechanisms and cell ion distribution of a broad range of cells, a concentration of agent is selected such that the treatment solution interferes selectively with the cellular mechanisms of lens epithelial cells while leaving other ocular cells substantially unharmed. The treatment solution selectively induces cellular death and/or detachment of lens epithelial cells while other ocular cells and tissue remain substantially unharmed and without lengthy preoperative pre-treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/244,878 filed Sep. 17, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

TECHNICAL FIELD

The present invention relates to novel treatment solutions and methodscomprising an ion transport mechanism interference agent, alone or incombination with other agents, used to prevent posterior capsularopacification by selectively inducing detachment and/or cell death oflens epithelial cells without damaging other ocular cells and tissue andwithout lengthy preoperative treatment.

BACKGROUND OF THE INVENTION

The predominant role of the lens of the human eye is to focus light raysthat have passed through the cornea and aqueous humour onto the retina.The structure and metabolism of the lens contributes directly towardmaintaining its integrity and transparency. The lens is composedentirely of epithelial cells in different stages of maturation and isrelatively unusual in that tissue is never discarded during thematuration process. As new lens cells are formed, older cells aredisplaced toward the interior of the lens. The lens soon becomesisolated from a direct blood supply and depends on the aqueous andvitreous humors for both nutrition and elimination pathways. The opticalcharacteristics of the lens are much dependent on lens cells maintaininga constant cell volume and dense packing of the fibers to reduce thevolume of intercellular space. Maintaining its delicate structuretherefore becomes an essential characteristic of the lens. The lens hasevolved its unique capabilities to maintain constant cell volume byregulating its ion, sugar, amino acid, and water balances.

A cataract of the human eye is the interruption of the transmission oflight by loss of lens transparency. Cataracts, which cause blurring andclouding of vision, are by far the most common cause of low visualacuity. The clouded lens can be removed by surgical procedure, i.e.extra-capsular cataract extraction (ECCE). ECCE comprises the removal ofthe clinical nucleus with cortical cleanup using either manual orautomated vacuuming techniques. The posterior and equatorial capsule isleft intact as an envelope or bag into which a posterior chamberintraocular lens can be inserted. If the posterior capsule and zonulesare intact, this lens will ordinarily remain in place throughout thepatient's life without any complications. During the operation, theanterior portion of the lens capsule is carefully opened and thecataract is removed. The intraocular lens is inserted into the remaining(posterior) portion of the capsule. This also results partially in aloss of natural lens accommodation.

Standard cataract surgery employs a procedure known asphaco-emulsification. This process agitates the lens content causingbreak-up of the lens material, which is then sluiced out of the lenscapsule by the phaco-emulsification probe that simultaneously injectsand extracts a washing solution. Both dead and live lens epithelialcells detached by the treatment will be washed out by this sluicingaction.

Progress is being made in the development of new treatments that involveretention of the anterior lens capsule and the replacement of the lenscontent with injectable material.

After this surgery, vision is restored but one of the most frequentcomplications of prevailing cataract surgery is the proliferation oflens epithelial cells (LECs) after cataract surgery. Posterior CapsularOpacification (PCO), also known as secondary cataract, is the mostfrequent complication following extra-capsular surgery, occurring inabout twenty to forty percent of patients. In the past decade, resultsfrom a number of experimental and clinical studies have led to a betterunderstanding of the pathogenesis of PCO.

The main cause of PCO is the proliferation and migration of residualLECs to the posterior lens capsule. Despite the care taken by surgeonsto remove most of the residual lens epithelial cells, they are difficultto remove. Many LECs are, therefore, left behind in the lens capsularbag at the end of the surgical procedure. The proliferation of the LECscauses the membrane or envelope into which the intraocular lens isplaced to become cloudy over time. Proliferation of LECs is also asignificant problem in the new cataract treatments utilizing, forexample, injectable lenses. The cloudy membrane is called an “aftercataract” or PCO. The symptoms of PCO are identical to those ofcataract, causing vision to gradually fade and eventually leading toblindness if not treated.

Much effort has been made to prevent or minimize formation of PCO. Theseefforts can be broadly categorized into three areas: surgicalimprovement, lens design improvement and chemical prevention.

A number of surgical strategies have been developed to attemptprevention of PCO. These have involved the use of various surgicalinstruments and the application of laser, ultrasound, and freezingtechniques. Once PCO has occurred, YAG laser capsulotomy is a simple,quick procedure in which a laser beam is used to create an opening inthe center of the cloudy capsule. There are, however severe risksassociated with YAG laser capsulotomy. These mainly include thedevelopment of retinal detachment, glaucoma, cystoid macula oedema.

Much interest has centered on the type of material from which theintraocular lens is made and the profile of the intraocular lens' edge.Biconvex and planer convex polymethylacrylate as well as silicone platehepatic intraocular lenses and lenses with sharp optic edges arereported to have a beneficial effect on PCO. Significant advances havebeen made in this area particularly by Alcon's Acrysof lens. Theselenses are however relatively expensive and introduce complications orconstraints of their own.

There have been other attempts to destroy or prevent proliferation ofLECs making use of chemical agents. In British Patent Application0122807.1, filed on Sep. 21, 2001, the hypothesis was that by decreasingintracellular sorbitol concentration in LECs by modulating sorbitolpathways via an aldose reductase inhibitor, PCO could be prevented byinducing death of LECs by osmotic shock. However, in order to modulatethe sorbitol pathways, the proposed treatment involved pretreating thelens capsular bag for a period of up to 48 hours prior to surgery andis, therefore, not easily incorporated into current cataract surgery.

Other chemical therapies have been attempted. For example, the use ofimmunotoxin-conjugated antibody specific for LECs can reduce but notcompletely prevent the incidence of PCO. See Clark et al, J. CataractRefract. Surg. 1998 December; 24(12): 1614-20 and Meacock et al, J.Cataract Refract. Surg. 2000 May; 26(5): 716-21. The use ofethylenediamine tetraacetic acid, Trypsin and DISPASE® (NeutralProtease) has also been used to separate the LECs but can damage thezonules and surrounding tissues. See Nishi, J. Cataract Refract. Surg.1999 January; 25(1): 106-17 and Nishi et al, Opthalmic Surg. 1991August; 22(8): 444-50. Other agents that have been tested for theprevention of PCO include RGD peptides to inhibit the migration andproliferation of lens epithelial cells; anti-mitotic drugs, such asMitomycin C and 5-fluorouracil. See Chung H S, Lim S J, Kim H B. JCataract Refract Surg. 2000 October; 26(10): 1537-42; Shin D H, Kim Y.Y., Ren J. et al. Ophthalmology. 1998; 105: 1222-1226.

Unfortunately most chemical agents described above, when used at theireffective doses, demonstrate unacceptable levels of toxicity tosurrounding ocular tissues including ocular cells such as cornealendothelial cells (CEDCs) and retinal pigmented epithelial cells(RPECs).

BRIEF SUMMARY OF THE INVENTION

In accordance with the present invention, the disadvantages of priormeans of preventing PCO as discussed above, have been overcome. Thepresent invention prevents PCO by rapidly and selectively inducingdetachment and/or cell death of lens epithelial cells (LECs) withoutsignificantly damaging other ocular cells and tissues, is easilyincorporated into existing standard cataract surgery and into futurepotential new treatments, and allows for easy removal of residual lensepithelial cells during or after cataract surgery. PCO prevention isaccomplished via application of treatment solution or solutions. Thetreatment solution is applied or introduced into the lens capsular bagbefore, during, or after cataract surgery. Alternatively, the treatmentsolution may be applied to an injectable intraocular lens prior tocataract surgery. The treatment solution comprises an ion transportmechanism interference agent, which either alone or in combination withother treatment agents such as an osmotic stress agent and an agent toestablish a suitable pH, selectively induces detachment and/or death oflens epithelial cells such that posterior capsular opacification isprevented. While the ion transport mechanism interference agent iscapable of interfering with the cellular mechanisms and cell iondistribution of a broad range of cells, a concentration of agent isselected such that the treatment solution interferes selectively withthe cellular mechanisms of lens epithelial cells while leaving otherocular cells substantially unharmed. The treatment solution selectivelyinduces cellular death and/or detachment of lens epithelial cells whileother ocular cells and tissue remain substantially unharmed and withoutlengthy preoperative pre-treatment.

The treatment solution or solutions, which may be applied or introducedbefore, during, or after cataract surgery, may selectively and rapidlyremove the LECs by: (i) inducing selective detachment of the LECs fromtheir substrates or membranes through interference with normal cellularfunctions, causing eventual cellular death or at least allowing for easyremoval of LECs; (ii) inducing selective death of LECs throughinterference with normal cellular functions; and/or (iii) inducingsusceptibility of the LECs to osmotic stress through interference withnormal cellular functions, ultimately leading to cellular detachmentand/or death. The present invention allows for chemical agents thatinterfere with the ion transport mechanisms of a broad range of cells tospecifically and uniquely target the LECs.

In one embodiment of the present invention, the ocular treatmentsolutions and methods prevent PCO by utilizing an ion transportmechanism interference agent to induce substantially greater incidenceof detachment and/or death of LECs than other ocular cells. The iontransport mechanism interference agent of the present invention iscapable of interfering, either directly or indirectly, withintracellular, extracellular and/or intercellular mechanisms thatregulate the flow of ions and water across the cellular membrane of aLEC. This interference may cause cells to shrink and/or swell, resultingin cell detachment and/or death. A concentration of agent is selectedsuch that, while the treatment solution comprising the ion transportmechanism interference agent causes significant detachment and/or deathof lens epithelial cells, it does not cause the death and/or detachmentof a significant percentage of other ocular cells. More particularly,the ion transport mechanism interference agent may include aco-transport interference agent, a sodium pump interference agent, anexchange interference agent, or a channel interference agent.

The ion transport mechanism interference agents may themselves inducecellular volume changes and/or cell detachment and/or death of LECs ormay sensitize LECs such that the LECs are rendered incapable ofsufficiently responding to an additional agent that causes osmoticstress, selectively detaching and/or killing the LEC and preventing PCO.This additional agent is an osmotic stress agent such as an osmolytethat induces osmotic shock in the LECs causing cell detachment and/ordeath.

The treatment solutions of the present invention have a suitable pHselected according to a selective concentration of a particular osmoticstress. The pH of the solutions can be adjusted by any acids, bases orother agent that increases or decreases the concentration of hydrogenions in the solution.

These and other advantages and novel features of the present invention,as well as details herein, will be more fully understood from thefollowing description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a diagram illustrating the cell viabilities of differentocular cells (LECs, RPECs and CEDCs) after solutions comprising varyingconcentrations of NaCl are introduced;

FIG. 2 is a diagram illustrating cell viabilities of LECs afterapplication of increasing concentrations of NaCl in the absence(diamonds) and the presence (circles) of 30 μM frusemide, a co-transportinterference agent;

FIG. 3 is a diagram illustrating cell viabilities of total LECs afterapplication of the solutions comprising increasing concentrations ofNaCl at varying pH levels;

FIG. 4 is a diagram illustrating cell viabilities of RPECs afterapplication of solutions comprising increasing concentrations of NaCl atvarying pH levels;

FIG. 5 is a diagram illustrating cell viabilities of LECs and RPECsafter application of treatment solutions comprising ≦200 mM NaCl,increasing concentrations of a co-transport interference agent,frusemide, and 311 of 5N NaOH;

FIG. 6 is a diagram illustrating cell viabilities of LECs, RPECs, andCEDCs after application of treatment solutions comprising ≦200 mM NaCl,increasing concentrations of a co-transport interference agent,bumetanide, and 3 μl of 5N NaOH;

FIG. 7 is three photographs illustrating the effects of treatment onprimary cultured LECs with the treatment solutions of the presentinvention at (A) zero, (B) five and (C) ten minutes after washing;

FIG. 8 is four photographs illustrating the effects of treatment onprimary LECs in a human organ culture PCO model with the treatmentsolutions of the present invention (A) before treatment, (B) fiveminutes after treatment, (C) ten minutes after treatment, and (D) afterwashing.

FIG. 9 is four photographs illustrating the effects of treatment onRPECs with a treatment solution comprising ≦200 mM NaCl, 90 μMfrusemide, and 3 μl of 5N NaOH at (A) zero, (B) five, and (C) tenminutes post-treatment and (D) after washing;

FIG. 10 is a diagram illustrating cell viabilities of LECs, RPECs andCEDCs after application of treatment solutions comprising ≦200 mM NaCl,90 μM frusemide and 3 μl of 5N NaOH;

FIG. 11 is photographs illustrating the effects of treatment onLECs-CCCs with a treatment solution containing 200 μM DHCT andhyperosmotic ≦200 mM NaCl at (A) zero minutes, (B) about two minutes and(C) after wash.

FIG. 12 is photographs illustrating the effects of treatment on in vivorabbit eyes with a treatment solution containing DHCT and hyperosmoticNaCl in (C, D, E) treated eyes three weeks after surgery compared to (A,B) control eyes three weeks after surgery.

FIG. 13 is photographs illustrating the effects of treatment onLECs-CCCs with a treatment solution containing 308 μM bumetanide andhyperosmotic NaCl at (A) zero minutes, (B) five minutes and (C) afterwashing.

FIG. 14 is photographs illustrating the effects of treatment on in vivorabbit eyes with a treatment solution containing bumetanide andhyperosmotic NaCl on treated rabbit eyes.

FIG. 15 is photographs illustrating the effects of treatment with atreatment solution containing NEM for human LECs at (A) zero minutes,(B) about three minutes and (C) two minutes after wash; and (D, E) on invivo rabbit eyes.

FIG. 16 is photographs illustrating the effects of treatment onLECs-CCCs with a treatment solution containing gramicidin and PBS at (A)zero minutes (B) two minutes and (C) after wash.

Table 1 is a table relating to the effects of different treatmentsolutions in the prevention of PCO in in vivo rabbit eyes.

DETAILED DESCRIPTION OF THE INVENTION

While the invention will be described in connection with one or morepreferred embodiments, it will be understood that the invention is notlimited to those embodiments. On the contrary, the invention includesall alternatives, modifications, and equivalents as may be includedwithin the spirit and scope of the claims concluding this specification.

The present invention prevents PCO by rapidly and selectively inducingdetachment from the lens capsule and/or death of lens epithelial cellsvia an ion transport mechanism interference agent, either alone or incombination with other agents, without substantially damaging otherocular cells and tissue and without lengthy pre-operative treatment.This selective detachment and/or death is made possible by the specificnature of LECs, which differentiates them from other cells in the eye.The result is that, at the doses and concentrations of agents used, LECsexperience a change in cell volume (e.g., shrink), are detached, killed,and/or removed from the lens, whilst other ocular cells and tissue areleft unharmed.

Water is effectively in thermodynamic equilibrium across the plasmamembrane of human cells. Because cell membranes of virtually all humancells are highly permeable to water, cell volume is determined by thecellular content of osmotically active solutes (electrolytes andosmolytes) and by the osmolarity and/or tonicity of the extracellularfluid. Generally, intracellular and extracellular tonicity are the same;under physiological conditions, most mammalian cells are not exposed tolarge ranges of tonicities. The osmolarity of body fluids is normallyabout 285 mOsm/kg H₂O and is regulated within extremely narrow limits(±3%) by body fluid homeostasis. However, under pathophysiologicalconditions, disturbances of body fluid homeostasis can be encountered.Plasma fluid osmolarities ranging between about 220 mOsm/kg H₂O andabout 350 mOsm/kg H₂O have been observed. Under these extreme conditionsthe cells of the body would, in the absence of volume-regulatingmechanisms, swell and shrink by twenty to thirty percent, respectively.

Lens epithelial cells, like other cells, use cell volume regulatoryprocesses to defend against changes in extracellular osmolarity and tomaintain constant cell volume. Volume regulatory mechanisms for lensepithelial, fibre and ciliary epithelial cells have been describedearlier. See for example, C. W. McLaughlin et al, Amer. J. Phsiol. CellPhysiol. 2001 September; 281 (3): C865-75; J. J. Zhang et al, Exp.Physiol. 1997 March: 82(2): 245-59; J. J. Zhang et al, Exp. Physiol.1994 September: 79(5): 741-53; J. J. Zhang et al, J. Physiol. 1997 Mar.1; 499(Pt. 2): 379-89. After osmotic perturbation, there ensues a volumeregulatory phase in which cells tend to return to the volume they had inan isotonic medium. Changes in cell volume activate specific metabolicand membrane transport pathways that result in the net accumulation orloss of osmotically active solutes. Regulatory volume decrease (RVD) isthe process used to decrease or maintain cell volume in response to lowextra-cellular osmotic pressure or tonicity. Regulatory volume increase(RVI) is the process used to increase or maintain cell volume inresponse to high extra-cellular osmotic pressure or tonicity. When cellsolute content (i.e., intracellular tonicity) or extracellular tonicityis altered, rapid transmembrane water flow occurs to restoreequilibrium. Because the plasma membrane is highly compliant, water flowcauses cell swelling or shrinkage.

One way that cells alter their intracellular ion concentrations toestablish equilibrium is through the use of ion transport mechanismslocated on the surface of the cells. These mechanisms move ions into orout of cells, altering intracellular ion strength and, as a result,osmotic pressure. For example, the ion transport mechanisms activatedduring RVD in various cell types involve conductive K⁺ and Cl⁻ channels(separate, conductive K⁺ and Cl⁻ transport pathways), K⁺—Cl⁻co-transport and functionally coupled K⁺—H⁺ and Cl⁻—HCO₃ ⁻ exchange. Theion transport mechanisms activated during RVI in various cell typesinvolve Na⁺—K⁺-2Cl⁻ co-transport, and functionally coupled Na⁺—H⁺ andCl⁻—HCO₃ ⁻ exchange.

There is a surprising diversity between different cell types in thenature and reaction of ion transport mechanisms. See Hoffmann andSimonsen, Physiol Rev. 1989 April; 69 (2): 315-82. For example, thereare no bumetanide-dependent ion cotransport mechanisms found in vivocorneal endothelium, but high expressions in lens epithelium. Haas, M.1994 Am. J. Physiol. 267, C869-C885; Lawrence et al 2001, Exp. Eye Res.73, 660. There is also a significant difference in volume set pointsamong different cells. For example, cells have certain volume set-pointsat which the cells capable of undergoing RVD and RVI are doing neither.Different cells have different volume set-points at which giventransport mechanisms turn on or off. See Parker, J. C Am J. Physiol.1993, 265: C1191-1200; Parker J C et al. J Gen Physiol. 1995 June;105(6): 677-99. The set point or threshold at which lens cells turn offtheir RVI and RVD processes is different from other ocular cells. Cellshape and interaction of integrins with the extra-cellular matrix play acritical role in determining cell detachment, survival or inducingprogrammed cell death in adherent cells. Volume regulation mechanismsare also essential for cell division. Additionally, the co-transportmechanisms may also be found in different locations in different ocularcells which may affect the outcome when a treatment is applied in theanterior chamber. For example, the cotransports in ciliary epithelium donot directly expose to the anterior chamber. (Jacob and Civann, Am. J.Physiol. 271 (40): C703-720, 1996.) These differentiate the LECs fromother cells in the eye and the body.

Interfering with the ion transport mechanisms of lens epithelial cellsprevents these cells from adequately responding to a hypoosmotic orhyperosmotic extra-cellular environment or other cellular stresses. Thefailure of the cells to respond to these stresses triggers immediate oreventual cell detachment and/or cell death.

Lens epithelial cells that are unable or rendered incapable ofresponding to stresses may have several responses. One response is celldeath. Another possible response is detachment of the cell from itsunderlying substrate or membrane. For example, the cell may shrivel suchthat the cell can no longer maintain its attachment to the substrate,which may be associated with rearrangement of the cell cytoskeleton.Cell shrinkage may also trigger adherent cells to rearrange their focaladhesion contacts and cytoskeletal attachments to those contacts. Thedetachment ultimately causes cellular death and/or allows for easyremoval of the cell. Cell death may lead to cell detachment fromsurrounding cells and the extra-cellular matrix (ECM) and celldetachment may occur without immediate cell death. For example,disruption of extracellular matrix contacts has been shown to induceanoikis, a form of apoptosis induced by cell detachment from itssubstation cells. Another possible response by lens epithelial cells isa weakening or sensitizing of the cell, such that the cell becomesvulnerable to osmotic stress or shock. For example, once a cell issensitized, it is rendered incapable of appropriately responding toosmotic shock or stress such that introduction of shock or stressors tothe sensitized cell will cause cell volume changes, detachment and/orcellular death. As shown in some examples, LEC volume changes,detachment and/or death after treatment may be evidenced by viewing thecellular area (or by other techniques including optical imagingtechniques, light and epifluorescence microscopy, confocal microscopy,electron microscopy, immunofluorescence techniques, in situhybridization techniques and techniques for protein identification andanalysis) before or after the area has been rinsed, washed, or in anyway agitated such that detached and/or dead LECs may be removed. Thetreatment solutions and methods of the present invention prevent PCO byselectively and rapidly eliciting at least one of these responses fromLECs while leaving other ocular cells substantially unharmed.

In one aspect, the ocular treatment solutions and methods of the presentinvention prevent PCO by inducing substantially greater incidence ofdetachment of the LECs from their substrates or membranes and/or celldeath than other ocular cells such as corneal endothelial cells (CEDCs)and retinal pigmented epithelial cells (RPECs) via an ion transportmechanism interference agent. Therefore, a significant advantage of thepresent invention is that treatment with the agents of the presentinvention prevents PCO by allowing for removal from the lens capsularbag of a substantial percentage of LECs, while other ocular cells andtissue are not significantly harmed.

The ion transport mechanism interference agent is an agent capable ofinterfering, either directly or indirectly, with intracellular,extracellular and/or intercellular mechanisms that regulate the flow ofions and water across or within the cellular membrane of a lensepithelial cell. The ion transport mechanism interference agentinterferes with normal cellular ion distribution mechanisms andconsequently the functions of cells. The ion transport mechanisminterference agent of the present invention, either alone or incombination with other agents, disturbs the normal distribution of LECcell ions and thereby selectively induces, either directly orindirectly, LEC detachment and/or death to prevent PCO. A concentrationof the agent is selected such that the treatment solution interfereswith ion transport mechanisms of LECs causing cellular volume changes,detachment and/or death of LECs, but does not substantially interferewith ion transport mechanisms of other ocular cells. While LECs may beeasily removed from the lens capsular bag due to cell detachment and/ordeath, other ocular cells remain substantially unharmed.

The ion transport mechanism interference agent is capable of interferingwith one or more of the following cellular mechanisms in an LEC: (1)co-transport mechanisms (for example, K⁺—Cl⁻ co-transport, Na⁺—Cl⁻co-transport, Na⁺—K⁺-2Cl⁻ co-transport and amino acid transport); (2)the sodium pump; (3) ion exchanges (for example, functionally coupledNa⁺—H⁺ and Cl⁻—HCO₃ ⁻ exchanges; functionally coupled K⁺—H⁺ and Cl⁻—HCO₃⁻ exchange, Cl⁻—Cl⁻ exchange, Ca²⁺—Na⁺ exchange); and (4) ion channels(for example, potassium channels, chloride channels, volume-sensitiveorganic osmolyte and anion channel (VSOAC), pore-forming proteins andpeptides and other anion channels). The ion transport mechanisminterference agent may activate or inhibit either directly or indirectlythese cellular mechanisms.

In hypotonic media, vertebrate cells initially swell by osmotic waterequilibration but subsequently regulate their volume by a net loss ofKCl and a concomitant loss of cell water to restore normal cell volume.Cell swelling activates transport pathways that result in the net effluxof potassium, chloride and organic osmolytes. The ion transportmechanisms activated during RVD in various cell types involve conductiveK⁺ and Cl⁻ channels (separate, conductive K⁺ and Cl⁻ transportpathways), K⁺—Cl⁻ co-transport, and functionally coupled K⁺—H⁺ andCl⁻—HCO₃ ⁻ exchange. For example, potassium and chloride are lost fromthe cell primarily through activation of the K⁺—Cl⁻ co-transport orthrough separate potassium and anion channels.

In contrast, in hypertonic media, vertebrate cells initially shrink byosmotic water equilibration but subsequently regulate their volume by anet gain of KCl and a concomitant uptake of cell water to restore normalcell volume. Cell shrinkage activates transport pathways that result inthe net influx of chloride, sodium, and potassium. The ion transportmechanisms activated during RVI in various cell types involve Na⁺—Cl⁻ orNa⁺—K⁺-2Cl⁻ co-transport, and functionally coupled Na⁺—H⁺ and Cl⁻—HCO₃exchange.

Examples of ion transport mechanism interference agents include, but arenot limited to:

-   -   n-ethylmaleimide    -   valinomycin    -   gramicidin    -   catecholamines    -   calcium    -   Ionophore A23187 (including, for example, Ionophore A23187 plus        calcium)    -   calmodulin    -   pimozide    -   loop diuretics (e.g., frusemide, bumetanide, ethacrynic acid,        piretanide, torasemide)    -   thiazide diuretics (e.g., dihydrochlorothiazide;        hydrochlorothiazide; cyclopenthiazide; benzthiazide;        chlorothiazide; bendroflumethiazide; chlorthalidone;        hydroflumethiazide; methyclothiazide; metolazone; polythiazide;        quinethazone; trichlormethiazide)    -   amiloride    -   di-isothiocyano-disulfonyl stilbene (DIDS)    -   staurosporine (SITS)    -   niflumic acid    -   stilbene derivatives (e.g. 4,4′-dinitrostilbene-2′2-disulfonic        acid)    -   quinine    -   Cytochalasin B

In general there are four classes of ion transport mechanisminterference agents: (1) co-transport interference agents; (2) sodiumpump interference agents; (3) exchange interference agents; and (4)channel interference agents. As reflected below, it is understood thatan ion transport mechanism interference agent may fall into one or moreof the four preceding classes.

A co-transport interference agent is an agent capable of interfering,either directly or indirectly, with the co-transport mechanisms of acell. More particularly, the co-transport interference agent activatesor inhibits the co-transport mechanisms of an LEC. A concentration ofco-transport interference agent is selected such that the treatmentsolution induces substantially greater incidence of detachment and/ordeath of LECs than other ocular cells. Examples of co-transportmechanisms include the K⁺—Cl⁻ or Na⁺—K⁺-2Cl⁻ cotransports. Examples ofco-transport interference agents include:

Diuretics, for example:

-   -   (i) thiazide diuretics such as Dihydrochlorothiazide,        Hydrochlorothiazide, Cyclopenthiazide, Benzthiazide,        Chlorothiazide, Bendroflumethiazide, Chlorthalidone,        Hydrochlorothiazide, Hydroflumethiazide, Methyclothiazide,        Metolazone, Polythiazide, Quinethazone, and Trichlormethiazide.        Commonly used brand names in the United States include:        Aquatensen (methyclothiazide), Diucardin (hydroflumethiazide),        Diulo (metolazone), Diuril (chlorothiazide), Enduron        (methyclothiazide), Esidrix (hydrochlorothiazide), Hydro-chlor        (hydrochlorothiazide), Hydro-D (hydrochlorothiazide),        HydroDIURIL (hydrochlorothiazide), Hydromox (quinethazone),        Hygroton (chlorthalidone), Metahydrin (trichlormethiazide),        Microzide (hydrochlorothiazide), Mykrox (metolazone), Naqua        (trichlormethiazide), Naturetin (bendroflumethiazide), Oretic        (hydrochlorothiazide), Renese (polythiazide), Saluron        (hydroflumethiazide), Thalitone (chlorthalidone), Trichlorex,        (trichlormethiazide), Zaroxolyn (metolazone), Apo-Chlorthalidone        (chlorthalidone), Apo-Hydro (hydrochlorothiazide), Diuchlor H        (hydrochlorothiazide), Duretic (methyclothiazide), HydroDIURIL        (hydrochlorothiazide), Hygroton (chlorthalidone), Naturetin        (bendroflumethiazide), Neo-Codema (hydrochlorothiazide),        Novo-Hydrazide (hydrochlorothiazide), Novo-Thalidone        (chlorthalidone), Uridon (chlorthalidone), Urozide        (hydrochlorothiazide), and Zaroxolyn (metolazone)    -   (ii) loop-acting diuretics such as benzmetanide, bumetanide,        torsemide, ethacrynic acid, and frusemide (also known as        furosemide). Commonly used brand names include: Bumex        (bumetanide), Demadex (torsemide), Edecrin (ethacrynic acid),        Lasix (furosemide), Myrosemide (furosemide), Apo-Furosemide        (furosemide), Edecrin (ethacrynic acid), Furoside (furosemide),        Lasix (furosemide), Lasix Special (furosemide), Novosemide        (furosemide), and Uritol (furosemide)    -   (iii) potassium-sparing diuretics. Communly used brand names in        the United States include as Aldactone (spironolactone),        Dyrenium (triamterene), and Midamor (amiloride). Commonly used        brand names in Canada: Aldactone (spironolactone), Dyrenium        (triamterene), Midamor (amiloride), and Novospiroton        (spironolactone))    -   (iv) carbonic anhydrase inhibitors such as Acetazolamide,        Dorzolamide and Brinzolamide    -   (v) diuretics with potassium such as Burinex K® (Leo), Centyl        K®; Lasikal®, and Neo-NaClex-K®    -   (vi) combined diuretics such as Triam-co [co-triamterzide        50/25]; Amil-co [co-amilozide 5/50], co-amilozide 5/50        [co-amilofruse 5/50], Moduretic [co-amilozide 5/50], Dyazide        [co-triamterzide 50/25], Navispare [amiloride 2.5        mg+cyclopenthiazide 250 mcg], co-amilofruse 5/40 [co-amilofruse        5/40] Fru-co [co-amilofruse 5/40] Frusene [triamterene 50        mg+frusemide 40 mg], Kalspare [triamterene 50 mg+chlortalidone        50 mg]; Burinex A [amiloride 5 mg+bumetanide 1 mg] Lasoride        [co-amilofruse 5/40], Frumil [co-amilofruse 5/40] Lasilactone        [spironolactone 50 mg+frusemide 20 mg], Aldactide 50        [co-flumactone 50/50], and Dytide [triamterene 50        mg+benzthiazide 25 mg]; Co-flumactone [Spironolactone with        thiazides]).

Examples of co-transport interference agents also include:

-   -   co-transport interference agents that cause cell shrinkage by        promoting increase of KCl efflux via K—Cl co-transport include        DDS-NOH, Dihydroindenyl[oxy]alkanoic acid (DIOA), okadaic acid        (OA), A-23187, 1-ethyl-2-benzimidazolinone, YM934, cromakalim,        gem-dimethyl substituent:        9-(3,4-dichlorophenyl)-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione        (A-184208), charybdotoxin (ChTX), Prostaglandin E2,        staurosporine, K+ ionophore valinomycin, tumor necrosis factor α        plus cycloheximide, diazoxide, pinacidil and nicorandil, and        agents that increase intracellular levels of cAMP or cyclic GMP        (Nelson et al., 1995), and epoxides of arachidonic acid.    -   any stimuli that induces potassium and chloride and other ion        and osmolyte loss (for example, intracellular or extracellular        calcium depletion or elevation) by activation of K—Cl        co-transport mechanisms    -   n-ethylmaleimide (NEM) (including, for example, n-ethylmaleimide        plus rubidium)    -   iodoacetamide    -   mercury (Hg²⁺)    -   and any other agent capable of capable of activating or        inhibiting a co-transport mechanism of a lens epithelial cell.

A sodium pump interference agent is an agent capable of interfering,either directly or indirectly, with the Na/K/ATPase pump, known as thesodium pump, mechanism of a cell. More particularly, the sodium pumpinterference agent activates or inhibits the Na/K/ATPase pump of an LEC.For example, LECs contain impermanent, anionic macromolecules and thecolloid-osmotic swelling, resulting from entry of diffusible ions andwater (Gibbis-Donnan equilibrium), is constantly counteracted by theoperation of ion-extruding pumps that extrude ions at a rate equal tothat of the dissipative leak entry. A concentration of a sodium pumpinterference agent is selected such that the treatment solution inducessubstantially greater incidence of detachment and/or death (e.g., viaswell) of LECs than other ocular cells. An example of a sodium pumpinterference agent includes ouabain and any other agent capable ofactivating or inhibiting a sodium pump of a lens epithelial cell.

Yet another type of ion exchange mechanism interference agent is anexchange interference agent. An exchange interference agent is an agentcapable of interfering, either directly or indirectly, with the ionexchanges of a cell. More particularly, the exchange interference agentactivates or inhibits the ion exchange mechanisms of a LEC, which notonly results in intracellular and extracellular ion re-distribution butmay also alter intracellular pH. Examples of ion exchange mechanismsinclude K⁺—H⁺ and Cl⁻—HCO₃ exchanges and Na⁺—H⁺ exchange functionallycoupled to Cl⁻—HCO₃ exchange. For example, any stimuli that inducepotassium and chloride loss by activation of the K⁺—H⁺ and Cl⁻—HCO₃ ⁻exchanges of an LEC is an exchange interference agent. Examples ofexchange interference agents include:

-   -   staurosporine (SITS)    -   amiloride    -   di-isothiocyano-disulfonyl stilbene (DIDs)    -   calmodulin (CaM)    -   copper (Cu²⁺)    -   hydrogen (H⁺)    -   and any other agent capable of activating or inhibiting an        exchange of a lens epithelial cell.

A channel interference agent is an agent capable of interfering, eitherdirectly or indirectly, with the ion channels of a cell. Moreparticularly, the channel interference agent activates or inhibits theion channels of an LEC. Examples of ion channel include the potassium,chloride, sodium, and VSOAC and pore-forming proteins and peptideschannels or transport pathways. For example, any stimulus that inducespotassium chloride and organic osmolyte loss by activation of potassiumand chloride channels is a channel interference agent. A concentrationof a channel interference agent is selected such that the treatmentsolution induces substantially greater incidence of detachment and/ordeath of LECs than other ocular cells. Examples of channel interferenceagents include:

-   -   ketoconazole (KETO)    -   5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)    -   1,9,-dideoxyforskolin (DDF)    -   tamoxifen    -   verapamit    -   quinine    -   barium (Ba²⁺)    -   diphenylamine-2-carboxylate (DPC)    -   anthracene-9-carboxylic acid    -   indocrinone (M−196)    -   pimozide    -   CBIQ    -   EBIO, 1-ethyl-2-benzimidazolone    -   tamoxifen+ATP    -   Sulfhydryl oxidizing agents    -   inhibitors of protein phosphorylation    -   4-chloro-benzo[F]isoquinoline    -   β-CCM, ZK 93426    -   flumazenil    -   adenosine    -   sulfonylurea    -   arachidonic acid (AA)    -   phospholipase C (PLC)    -   protein kinase C    -   G-proteins    -   agents that induce changes in F-actin organization    -   and any other agent capable of activating or inhibiting an ion        channel of a lens epithelial cell.

Pore-forming proteins and peptides are the agents capable of creating apathway of a lens epithelial cell for molecules that cannot normallycross their lipid bilayer. More particularly, these PPFPs form a pore orchannel to promote intracellular K leak out from an LEC. Hundreds ofpeptide antibiotics have been described in the past half-centuryincluding gramicidin, bacitracin; polymyxin (B) (Neosporin, Otosporin),Nystatin (Nystan, Tri-Adcortyl), glycopeptides, a-defensin, b-defensin1, tachyplesin, bacterial nisin, a-hemolysin, roimmunolysins, Porins(OmpA, PhoE, NmpC, OmpF, Phosphoporin, LamB porin), Bcl-XL; α-hemolysin;Aerolysin, colicins, alamethecin, magainin, cecropin; amphiphilic βsheet peptides (gramicidin, defensin, and cytolysin), α-helical peptides(alamethecin, pneumolysin, magainin and cecropin), ranalexin, brevinin,and PR39.

Using the agents of the present invention, a concentration of agent oragents is selected such that application of the treatment solutionresults in LEC cell viability of preferably less than about ten percent,more preferably less than about five percent, and more preferablyapproaching zero percent. The desired cell viability for other ocularcells such as RPECs and CEDCs is preferably greater than about seventypercent, more preferably about ninety percent, and more preferablyapproaching one hundred percent.

Under these circumstances, the treatment solution comprising the agent,perhaps in combination with other agents, selectively induces LECsdetachment and/or death and induces substantially greater incidence ofdetachment and/or death of LECs than other ocular cells. As shown in theexamples, cellular detachment and/or death may be evidenced by viewingthe cellular area before or after the area has been rinsed, washed, orin any way agitated such that detached and/or dead LECs may be removed.

The agents of the present invention described above (referred tohereafter as the primary agent) selectively induce detachment and/ordeath of an LEC, directly or indirectly, alone or in combination withadditional agents described below or other agents and/or chemicals. Forexample, the ocular treatment solutions and methods of the presentinvention may comprise an osmotic stress agent, in addition to theprimary agent, wherein the solution induces substantially greaterincidence of cell detachment and/or death of lens epithelial cells thancorneal endothelial cells and retinal pigmented epithelial cells toprevent posterior capsular opacification.

An osmotic stress agent is an agent capable of inducing osmotic shock inan LEC. Osmotic shock in an LEC may be caused by any cellular conditionthat alters normal intracellular or extracellular ion equilibrium. Forexample, changes in the extracellular concentrations of osmoticallyactive solutes and various stimuli that affect the activity of thespecific ion transport systems involved in cell volume control (e.g.,hormones, growth factors and other external stimuli, the cell membranepotential, and cytoplasmic second messengers) may induce osmotic shock.Large transcellular ion fluxes may shock cellular volume homeostasis.Hypoosmotic stress can be caused by exposure of cells to a hypotonicsolution or a solution with a hypoosmotic osmolarity. Conversely,hyperosmotic stresses can be caused by exposure of cells to a hypertonicsolution or a solution with a hyperosmotic osmolarity. An increase ordecrease in the cellular concentration of osmotically active ions andorganic solutes may also shock cellular volume regulation and activatemembrane transport processes involved in the RVD or RVI response.

The application or introduction of an osmotic stress agent isappropriate where the primary agent sensitizes the LEC to osmotic shock(for example, by inhibiting an ion transport mechanism) and where theosmotic stress agent itself induces cell detachment and/or death. Forexample, if the ion transport mechanism interference agent inhibits anessential ion transport mechanism such as the sodium-potassium pump orco-transport, the LEC is incapable of defending itself against externalosmotic stresses or shocks, thereby enhancing its sensitivity toextracellular osmotic pressure caused by an additional osmotic stressagent. The failure of LECs to respond to osmotic stresses or shocks maytrigger cell death through activation of death-signaling processes.

However, it is understood that the primary agents may also be osmoticstress agents such that a primary agent may both selectively interferewith the ion transport mechanisms of LECs and induce osmotic shock.

More particularly, the osmotic stress agent has a hyperosmotic orhypoosmotic osmolarity. A hypoosmotic solution is one that has a lowersolute concentration than that normally found in physiologicalconditions; a hyperosmotic solution has a higher solute concentrationthan that normally found in physiological conditions. In other words,the osmotic stress agent creates extracellular hypertonicity orhypotonicity.

Any physiologically tolerable electrolytes and osmolytes, any solutethat contributes to osmotic strength, are suitable for inducing osmoticstresses or shocks. Examples of osmolytes include:

-   -   salts (such as NaCl) including:    -   (i) inorganic salts with cations including sodium, potassium,        lithium, rubidium, beryllium, magnesium, calcium, strontium,        barium, aluminum, zinc, tin, silver, gold, iron, mercury and        corresponding anions including chloride, bromide, iodide,        hydroxide, borate, metaborate, carbonate, hydrogen carbonate,        nitrate, fluoride, sulfate, monophosphate, diphosphate,        phosphate, thiocyanate and isothocyanate    -   (ii) organic salts with the same cationic ions as above and        corresponding anions such as acetate, alginate, benzoate,        butyrate, caprylate, caproate, citrate, decansulfonate,        decylsulfate, dodecylsulfate, diethyl barbiturate,        dimethyldithiocarbamate, dithionite, dodecansulfonate,        ethansulfonate, fluoroacetate, fluorophosphates, furmarate,        hexafluorophosphate, hexansulfonate, iodoacetate, L-lactat,        D-lactat, D,L-lactat, maleinate, malonate, mesolaxate,        myristate, oleate, oxalate, palmitate, propionate, salicylate,        tartrate, and trifluoro acetate;    -   amino acids (such as taurine, glycine, and alanine);    -   sugars (such as mannitol, myo-inositol, betaine,        glycerophosphorylcholine, betains, urea, sucrose and glucose);    -   and any other organic or inorganic osmolytes.

It is noted that while the osmolyte may be sodium chloride, as describedabove, the chloride may be replaced by iodide, bromide, nitrate,thiocyanate, fluoride, sulfate, isethionate, gluconate, and any otheracceptable substitute; similarly, sodium may be replaced by potassium,cesium, rubidium, lithium, francium, and any other acceptablesubstitute.

Preferred osmolytes are sodium chloride, potassium chloride, sodiumbromide, sodium citrate, sodium lactate, sodium hydroxide, sodiumiodide, sodium carbonate, sodium hydrogen carbonate, sodium nitrate,sodium fluoride, sodium sulfate, potassium carbonate, potassium citrate,potassium lactate, potassium hydrogen carbonate, potassium bromide,potassium hydroxide, potassium iodide, potassium nitrate, potassiumsulfate, cesium chloride, rubidium chloride and lithium chloride.

The osmotic stress agent and the primary agent may be appliedseparately, simultaneously or sequentially in the treatment of PCO.

The treatment solution may preferably have a pH from about 5 to 11,depending on the agents and concentrations used. In a preferredembodiment, a treatment solution comprising approximately ±200 mM NaCl,and more preferably ≦200 mM NaCl, has a pH preferably in the range ofabout 8 to 10, more preferably 7 to 10, and more preferably 7.6. The pHof the treatment solution may be adjusted by varying the concentrationsof the primary agents or the osmotic stress agent or by the introductionof an additional chemical agent that alters the pH. The pH can beadjusted by any acids or bases or any agent that increases or reducesthe hydrogen ion concentration.

The treatment will work with a range of concentrations of primaryagents, osmotic stress agents and agents which adjust pH. In accordancewith the present invention, suitable concentrations of agents areselected such that an agent that may be capable of interfering with thecellular mechanisms of a broad range of cells selectively interfereswith the ion transport mechanisms of LECs, causing LEC detachment and/ordeath. Application of the treatment solution results in a significantpercentage of LECs that are detached and/or killed whilst a lowpercentage of other ocular cells are harmed. At one end of the range ofconcentrations, the efficiency of the treatment solution is reduced,resulting in longer times to kill the LEC cells. At the other end of therange the medicament is prone to damaging other cells in the eye.Suitable concentrations of both the primary agent, the osmotic stressagent and the agents which adjust pH should be selected such that PCOmay be prevented by selective cell death and/or detachment of LECswithout significant damage to other ocular cells and ocular tissue.Preferably, concentrations of the agent or agents should be selectedsuch that, upon application, cell viability for LECs is less than aboutten percent, more preferably less than about five percent, and morepreferably approaching zero percent and the cell viability for otherocular cells such as RPECs and CEDCs is greater than about seventypercent, more preferably greater than about ninety percent, and morepreferably approaching one hundred percent. The concentration of activeingredients depends on the pharmacological activity of the activeingredients, but is generally preferably less than 1500 μM, morepreferably lower than 1000 μM, and more preferably lower than 500 μM.

The agents described above and the concentrations in which they areintroduced are interrelated. For example, a high pH treatment solutionwill kill LECs with lower levels of osmolarity. Examples of suitableconcentrations of certain agents are provided in the examples below.Moreover, the concentrations can be adjusted to suit the particularneeds of prevailing cataract surgery techniques and the needs of thepatient.

The treatment solution of the present invention may be administeredbefore, during, or after cataract surgery to prevent PCO. The anteriorportion of the lens capsule is opened and the cataract removed. Theintraocular lens may then be inserted into the posterior portion of thecapsule. Alternatively, the lens or lens content may be replaced with aninjectable intraocular lens (also known as lens refilling technique).The intraocular lens and lens materials may be pretreated with thetreatment solution of the present invention before it is placed into thelens capsule bag (e.g., by injection or insertion). The residual LECsmay or may not be removed using vacuuming or other surgical techniques.Phaco-emulsification, laser or other techniques may be employed toremove the lens contents. The treatment solution or treatment solutionsmay be applied or introduced topically at any time before, during orafter this process. The ocular region may then be washed to removeresidual cells.

As described above, the treatment solution may comprise the primaryagent, the osmotic stress agent, and any chemical agent used to adjustthe pH of the solution. Alternatively, separate solutions comprising theprimary agent, the osmotic stress agent, and/or the chemical agent toadjust pH may be applied or introduced either sequentially orsimultaneously to prevent PCO. As described above, the primary agent mayalso be an osmotic stress agent. The solution or solutions may beapplied via syringe, dropper, probe used for phaco-emulsification or anyother suitable or convenient means of application.

An additional advantage of the present invention is that the treatmentsolutions induce relatively rapid cell volume changes (e.g., shrinkage,detachment and/or death of lens epithelial cells such that the treatmentof the present invention is easily incorporated into cataract surgery.Detachment of lens epithelial cells may occur within about thirtyminutes of application of the solution or agents of the presentinvention, with cell death occurring within several hours. Preferably,cell volume changes and/or detachment occur within about fifteen minutesof application but is dependent on the concentrations and agentsselected.

Whether the treatment solutions and methods of the present inventionhave induced substantially greater incidence of cell detachment and/ordeath of LECs than other ocular cells or whether such treatment isselective may be determined by a comparative assessment of cellviability after treatment or any other acceptable method of determiningeffectiveness. More particularly, the treatment solutions and methodsshould prevent PCO while causing only minimal physiological damage toother ocular cells as described. Preferably, concentrations of the agentor agents should be selected such that, upon application, cell viabilityfor LECs is less than about ten percent and the cell viability for otherocular cells such as RPECs and CEDCs is greater than about seventypercent. It is understood that the treatment solution or solutions maycomprise additional agents, chemicals, proteins, and/or substances andmay comprise more than one primary agent or osmotic stress agent.Additional chemical or biological components may be added to improve theproperties of the treatment solution or solutions, for example,physiological tolerance, viscosity or storage capability.

The treatment solution may be used to coat an intraocular lens and/orintraocular lens materials prior to cataract surgery or may be mixedwith intraocular lens materials for use when a lens refilling techniqueis employed. The treatment solution may be packaged in kits or otherpackaging or stored either as a unitary solution or, as described above,in separate solutions for later application. For example, a kit orpackage may include a solution containing the ion transport mechanisminterference agent packaged separately from a solution containing theosmotic stress agent. Furthermore, the treatment solution may bepackaged in kits or packaging containing fluids used duringphaco-emulsification or in kits used for removal of lens content priorto injection of an injectable intraocular lens.

EXAMPLES Comparative Example 1

As described below, the confluent monolayers of LECs, CEDCs, and RPECsgrown in 96-well flat-bottom plates are exposed to varyingconcentrations of NaCl (an osmolyte) solution. Two hundred μl of NaClsolution (prepared as described below) are added to each well atconcentrations of 137 mM, 170 mM, 340 mM, 680 mM, 1360 mM and 2000 mMNaCl, respectively. The solutions have a pH of 8.0±0.4. Theconcentrations of NaCl correspond to final osmolarities of about 285 to4000 mOsm/L, respectively. Within about five to ten minutes afteraddition of hypertonic NaCl, some of the LECs began to shrink, round upand lose adhesion from the monolayer in a dose-dependent manner. Thiseffect is not seen as significantly in the CEDCs and RPECs.

Cell viabilities of LECs, CEDCs and RPECs in response to increasinghyperosmotic NaCl are shown in FIG. 1 (LECs shown in light graydiamonds, RPECS in dark gray squares, CEDCs in black diamonds). Eachpoint represents the mean of two experiments. At a hypertonic stress ofabout 1380 mM NaCl, about forty percent of the LECs survive while morethan eighty percent of CEDCs and RPECs survive, evidencing the differentosmotic tolerance between these cells.

The primary and first passages of human lens epithelial cells, humancorneal endothelial cells, and human retinal-pigmented epithelial cellsare cultured. The LECs, the CEDCs, and the RPECs are isolated frompost-mortem eyes by using standard techniques of cell isolation andenzyme digestion. See Uebersax E D, Grindstaff R D and Defoe D M Exp EyeRes. 2000 March; 70(3): 381-90; Wagner L M, Saleh S M, Boyle D J, andTakemoto D J. Mol Vis. 2002 Mar. 14; 8: 59-66; Cammarata P R, Schafer G,Chen S W, Guo Z, and Reeves R E. Invest Ophthalmol Vis Sci. 2002February; 43(2): 425-33; Rakic J M, Galand A, and Vrensen G F. Exp EyeRes. 2000 November; 71(5): 489-94. LECs and CEDCs are cultured inEagle's minimum essential medium (MEM). RPECs are cultured in Ham's F10medium, supplemented with 2 mM glutamine, 25 mmol/L Hepes, pH 7.4, 10U/mL penicillin, and 10 μg/mL streptomycin and 15% heat-inactivatedfoetal cow serum (FCS). The primary cultured LECs, CEDCs, and RPECs aresub-cultured at 10⁴ cells/wells in 96-well flat-bottom plates until theyreached 90 to 100% confluence. These cells are grown to confluence in anincubator with a humidified atmosphere of 5% CO₂ at 37° C. The cells arekept in a confluent state for overnight before assays.

Hyperosmotic solutions of NaCl are prepared by first making a 2 M NaClstock solution by adding 58.44 grams of NaCl to 500 ml dH₂O (NaClsupplied by Sigma Chemical co, Dorset, England). A desired concentrationof osmotic NaCl solution is then prepared by diluting the 2M stocksolution with distilled water (dH₂O). For example, to make ahyperosmotic solution of 170 mM NaCl, the 2M NaCl stock solution isdiluted with dH₂O at a ratio of 1 to 10.1; to make a hyperosmoticsolution of 340 mM NaCl, the 2M NaCl stock solution is diluted with dH₂Oat a ratio of 1 to 4.56. Final osmolarities (mOsm/kg/H₂O) are determinedby a freezing point depression method using a Cryoscopic Osmometry.

After treatment, the treated cells are thoroughly washed, and culturedwith fresh media for twelve hours before cell viability is assessed. Thecapability of these cells to survive at the indicated hypertonic stresslevels is assessed by an MTT assay 12-24 hours post-exposure.

MTT assay is an assay to assess mitochondria function, performed asdescribed by Mosmann (Mosmann, T. J Immunol Methods. 1983 Dec. 16;65(1-2): 55-63. The tetrazolium salt3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT;supplied by Sigma Chemical Co.) is dissolved in Phosphate's BalancedSalt (PBS) Solutions at 5 mg/ml and sterilized by passage through a 0.22μm Millipore filter. MTT stock solution is added to growth media at aratio of 1 to 10. Cells are incubated with this growth media for threeto four hours. In cells that are alive at the time of adding MTT,mitochondrial dehydrogenase cleaves the tetrazolium ring into a darkblue formazan reaction product, which builds up in the cell. MTT-reactedcultures are observed under brightfield microscopy without furtherprocessing. Cells in each well are scored as MTT-positive if they arecompletely filled with reaction product (in the case of unattachedcells) or contain many formazan crystals emanating from multiple foci(in the case of attached or spread cells). MTT-reacted cultures are thenlysised by dimethyl sulfoxide (DMSO) and read in a Microplate Reader toobtain a measure of cell viability. The number of live cells(MTT-positive) per well is determined at the specified concentrationsand expressed as a percentage of total cells (demonstrated in FIG. 1).

Example 2

Confluent monolayers of LECs are given three washes with Hank's BalancedSalt Solutions (HBSS) to remove FCS before the experiment. LECs areprepared in 96-well culture plates as in Example 1. These cells areexposed for about five to ten minutes to 200 μl/well of treatmentsolutions comprising increasing concentrations (˜135 mM, ˜170 mM, ˜340mM, ˜680 mM, ˜1360 mM and ˜2000 mM) of NaCl (an osmolyte) in thepresence and the absence of 30 μM frusemide (a co-transport interferenceagent, an ion transport mechanism interference agent). The treatmentsolutions have a pH of 8.0±0.4. After treatment, the treated cells arethoroughly washed, and cultured with fresh media. The viability of thetreated LECs was determined by MTT assay 12-24 hours post-treatment asdescribed in Example 1.

After this treatment, the same phenomenon of cell shrinkage anddetachment as observed in Comparative Example 1 also occurs in thepresence of frusemide, but the changes are much more dramatic. FIG. 2shows that in the absence of frusemide (depicted in diamonds) about 40%of LECs survive after a rapid hypertonic shock with 1380 mM NaCl.However, after inhibition of co-transport mechanisms using frusemide(depicted in circles), the osmotic tolerance of LECs is dramaticallyreduced, and almost no cells survive after rapid hypertonic shock with340 mM NaCl. This result shows survival capability of the LECs shiftsfrom hypertonic shock with 200 mM NaCl in the absent of frusemide to 170mM NaCl in the presence of the frusemide.

Each point represents the mean±standard deviation (S.D) of threeexperiments. A stock solution of 30.3 mM frusemide is prepared by firstdissolving 20 mg frusemide in 100 μl DMSO to make sure it dissolved andthen adding it to 1.9 ml of the desired concentration of hyperosmoticNaCl. To get 30 μM frusemide, five μL of frusemide stock solution isthen added to five ml of the desired concentration of NaCl solution(which is prepared as described in Comparative Example 1).

Comparative Example 3

This example demonstrates effects of pH on LECs osmotic survival.Confluent monolayers of the LECs are prepared as described inComparative Example 1. A control group of cells are exposed to asolution comprising physiological 135 mM NaCl at a pH of 7.4±0.3. Adifferent group of cells are exposed for ten minutes to 200 μl/well ofthe solutions comprising increasing concentrations of NaCl (˜170 mM, and340 mM) at varying pH (8.0±0.4, 8.4±0.5, 8.9±0.8, and 10.6±0.5).

The pH of the solutions is adjusted by directly adding 0, 1, 3, or 51 μlof 5N NaOH stock solution to 10 ml of the selected hyperosmotic NaClsolutions (which are prepared as described in Example 1). Thecorresponding final pH was determined by using a professional pH metre.Final pH in 10 ml of the NaCl solution without addition of NaOH gave areading of pH about 8.0±0.4. Final pH in 10 ml of the NaCl solution with111 of 5N NaOH gave a pH reading of about 8.4±0.5. Final pH in 10 ml ofthe NaCl solution with 3 μl of 5N NaOH gave a pH reading of about8.9±0.8. Final pH in 10 ml of the NaCl solution with 5 μl of 5N NaOHgave a reading of about 10.6±0.5.

Cell viability of the treated cells is determined by MTT assay 12-24hours post-treatment as described in Example 1. Cell viability of LECsafter application of solutions is depicted in FIG. 3. As shown, the graybar represents the control group in which LECs were exposed tophysiological 135 mM NaCl. The black and white bars represent the LECsexposed to 170 mM NaCl and 340 mM NaCl, respectively, with increasingpH. As shown, LECs sensitivity to hypertonic stress increases withincreasing pH and increasing NaCl concentrations. At pH of about10.6±0.5, about greater than seventy five percent of LECs burst and diewithin five minutes. At pH of about 8.9±0.8, the LECs detach effectivelyfrom their substrates but do not burst significantly.

Comparative Example 4

This example demonstrates effects of extracellular pH on the RPECsosmotic survival. Confluent monolayers of RPECs are prepared asdescribed in Example 1. These cells are exposed for ten minutes to 200μl/well of the solutions comprising increasing concentrations of NaCl(137 mM, 170 mM, 340 mM, 680 mM, 1360 mM and 2000 mM) at varying pH(8.4±0.5 and 10.6±0.5). The solutions of NaCl are prepared as describedin Example 1. The pH of the solutions is adjusted with additions of 1 μland 5 μl of 5N NaOH as described Example 3.

The viability of the treated cells was determined by MTT assay 12 to 24hours post-treatment as described in Example 1. Cell viability of RPECsafter application of the solutions is depicted in FIG. 4 (pH 8.4±0.5depicted in diamonds; pH 10.6±0.5 depicted in circles). Each pointrepresents the mean±standard deviation of three experiments. As shown,there is a dramatic decrease in the survival level of RPECs exposed tohypertonic NaCl when the pH level is raised.

Example 5

Confluent monolayers of LECs and RPECs are prepared as described inExample 1. These cells are exposed for about ten minutes to 200 μl/wellof treatment solutions comprising: (i) ≦200 mM hyperosmotic NaCl; (ii)increasing concentrations (10 μM, 30 μM, 60 μM, 90 μM, and 120 μM) ofco-transport interference agent, frusemide, and (iii) 3 μl of 5N NaOH.

The treatment solutions containing frusemide are prepared as describedin Example 2, by diluting stock solution of frusemide with ≦200 mMhyperosmotic NaCl solution (prepared as described in Example 1). Forexample, to get a hyperosmotic NaCl solution with 60 μM frusemide, 10 μLof frusemide stock solution is added to 5 mL hyperosmotic NaCl solution.The pH of the solutions is adjusted with 3 μl of 5N NaOH.

The viability of the treated cells is determined by MTT assay 12 to 24hours post-treatment as described in Example 1. Cell viability of LECs(circles) and RPECs (diamonds) are depicted in FIG. 5. Each pointrepresents the mean±standard error (s.e.m) of six experiments for theHLECs and four experiments for the HRPECs. As shown, at 10 μM frusemide,more than 50% of the LECs shrink, are lost or die. Between 30 μM and 120μM frusemide, about >90% of LECs are removed or killed. On the otherhand, between 30 μM and 90 μM frusemide, few RPECs are removed orkilled. Therefore, for example, in about 200 mM hyperosmotic NaClsolution at this pH, about 15 μM to about 100 μM are effectiveconcentrations at which no significant harm was done to RPECs.

Example 6

Confluent monolayers of LECs, CEDCs and RPECs are prepared as describedin Example 1. These cells are exposed for about ten minutes to 200μl/well of treatment solutions comprising: (i) ≦200 mM hyperosmoticNaCl, (ii) increasing concentrations (14 μM, 28 μM, 56 μM, 112 μM, and224 μM) of bumetanide, a co-transport interference agent, and (iii) 3 μlof 5N NaOH.

The treatment solutions containing Bumetanide are prepared as follows. Astock solution of 1.4 M bumetanide is prepared. A second stock solutionof 14 mM bumetanide is then made by diluting 1 μL of the 1.4 Mbumetanide solution with 1 mL hyperosmotic NaCl solution (prepared asdescribed in Example 1). The final desired concentrations of bumetanideare obtained by diluting the 14 mM bumetanide stock solution with NaClsolution to get concentrations of approximately 0 M (control) 14 μM, 28μM, 56 μM, 112 μM, and 224 μM, respectively. The pH of the solutions isadjusted with 3 μL of 5 N NaOH.

The viability of the treated cells was determined by MTT assay 12 to 24hours post-treatment as described in Example 1. FIG. 6 depicts cellviability of LECs (circles), RPECs (diamonds) and CEDCs (squares). Eachpoint represents the mean±s.e.m and standard deviations of sixexperiments for LECs and three experiments for RPECs and CEDCs,respectively. As shown, at 28 μM, 112 μM, and 224 μM bumetanide, nearlyall of the LECs shrink, detach and/or die under phase-contrastmicroscopic observation, and there are almost no cells left afterwashing. Nearly 100% cells show MTT negative response. On the otherhand, at these same concentrations, less than about 20% RPECs and 30%CEDCs are killed or removed. Accordingly, 28 μM, 112 μM, and 224 μMbumetanide are suitable therapeutic concentrations for these particulartreatment solutions.

Example 7

Confluent monolayers of LECs are prepared as described in Example 1 andgrown on a glass cover-slip instead of 96-well flat bottom cultureplates. The LECs are then thoroughly washed to remove serum.

Ten milliliters (10 mL) of the treatment solution containing ≦200 mMNaCl, 90 μM frusemide and 3 μl of 5N NaOH is prepared in a deep 35 mmpetri-dish.

The cover-slips are then transferred to the petri-dish containing thetreatment solution and are kept in the treatment solution for five toten minutes. The treatment solution is then poured away, and thecover-slips along with detached LECs are thoroughly rinsed with a freshHBSS solution with slight pressure using an aspiration/irrigation probe.After immediate examination and photography using inverted phasecontrast microscope, the LECs are kept in culture for seven days withfresh medium to examine whether any residual cells would survive.

FIG. 7 depicts images of LECs taken using an inverted phase-contrastmicroscope and their osmotic responses during treatment. Photograph 7Adepicts, at zero minutes, confluent LECs covering the entire area.Photograph 7B depicts, at five minutes post-treatment, the LECs roundedup and separating from their substrate. Photograph 7C depicts, at tenminutes post-treatment and after washing, a substantially clearsubstrate after the cells have been washed away.

Example 8

In order to assess the effects of the therapy in an environment close tothat in vivo, a lens organ culture PCO model is used (model wasoriginally developed by Liu et al (Liu C S, Wormstone I M, Duncan G,Marcantonio J M, Webb S F, Davies, P D. Invest Ophthalmol Vis Sci. 1996April; 37 (5): 906-14). Human lenses are isolated from donor eyes (agesbetween 19 to 89 years), and then glued onto a tailor-made stainlesssteel stand. Under an operating microscope, the anterior portion of thelens capsule 100, which is about 6 mm in diameter, is carefully openedusing the technique of continuous curvilinear capsulorhexis. The lensnucleus is then hydroexpressed with HBSS and the remaining lens fibrescarefully removed. The posterior capsule 200 and equatorial capsule 300are left intact as envelopes (bags), stored in plastic culture dishes(deep 35 mm petri-dish) and covered with Eagle's Minimum EssentialMedium growth medium (MEM) supplemented with 10% to 15% FCS. The humanLECs in the organ culture model are then allowed four to five days torecover from the surgical trauma.

Two groups of organ cultured PCO models were established. The firstgroup of ten lenses was established as a control and kept in organculture without treatment. All of these lenses developed confluent mono-or multiple-layered proliferated LECs on the anterior capsule 100 andposterior capsule 200 after about four to seven days.

The second group of twenty lenses, after four to five days of culture,also had a monolayer of the proliferated LECs. This group was thoroughlywashed to remove serum prior to the introduction of the treatmentsolutions. Ten milliliters (10 mL) of treatment solutions comprising:(i) ≦200 mM NaCl, (ii) about 90 μM frusemide, or about 28 μM bumetanide,or about 112 μM bumetanide, and (iii) 31 μl of 5N NaOH are prepared in adeep 35 mm petri-dish. The solutions are prepared as described in otherexamples. The organ cultures are then transferred to the petri-dishcontaining the treatment solutions and are kept in the treatmentsolution for five to ten minutes. The organ cultures are thentransferred using forceps into another petri-dish. The capsules alongwith detached LECs in the PCO organ culture are then thoroughly rinsedwith a fresh HBSS solution with slight pressure using anaspiration/irrigation probe. After immediate examination, the organcultures are kept in culture for seven days with fresh medium to examinewhether any residual cells would survive.

The effect of this treatment on LECs in PCO organ culture model isexamined on inverted microscope using phase contrast optics. They arephotographed with T-Max 400 film (Kodak) at zero, five, and ten minutesand at twelve hours post-treatment. As shown in FIG. 8, within five toten minutes of treatment, all of the LECs round up and lose adhesionfrom the monolayer and the lens capsule. Phase contrast micrograph 8Adepicts at low magnification (field of view is 1 mm×0.75 mm) theproliferated LECs after a sham ECCE operation on the anterior 100 andposterior capsule 200 six to seven days after dissection and beforetreatment. Phase contrast micrograph 8B depicts at high magnification(field of view is 3.4 mm×1.5 mm) the LECs starting to round up anddetach from the lens capsule five minutes after treatment. Phasecontrast micrograph 8C depicts at high magnification the majority oftreated cells detached from the monolayer ten minutes after treatment.Phase contrast micrograph 8D depicts at low magnification that, afterwashing, the lens capsule was devoid of LECs ten minutes aftertreatment.

Example 9

In this example, the effects of the treatment on primary RPECs areexamined. To assess whether there are any side effects on other ocularcells, the primary cultured RPECs were cultured in 6-well flat-bottomculture plate until they reached 90 to 100% confluence in Ham's F10medium, supplemented with 2 mM glutamine, 25 mmol/L Hepes, pH 7.4, 10U/mL penicillin, and 10 μg/mL streptomycin and 15% heat-inactivatedfoetal cow serum (FCS). The cultures were kept in an incubator with ahumidified atmosphere of 5% CO₂ at 37° C. and were kept in a confluentstate for overnight before assays.

The RPEC cultures were thoroughly washed to remove serum prior to theintroduction of the treatment solutions. Twenty milliliters (20 mL) ofthe treatment solution comprising (i) ≦200 mM NaCl, (ii) about 90 μMfrusemide, or about 28 μM bumetanide, or about 112 μM bumetanide, and(iii) 6111 of 5N NaOH is prepared in a deep 35 mm petri-dish asdescribed in previous examples. Five milliliters (5 ml) of the treatmentsolution is then added to the confluenced RPECs in each well of 3 wellsin a 6-well culture plate. After about 10 minutes, the solution isremoved completely and the treated RPECs are thoroughly rinsed with afresh HBSS solution with slight pressure using an aspiration/irrigationprobe. After immediate examination, the primary RPEC cultures are keptin culture for seven days with fresh medium to examine whether anyresidual cells would survive.

The effect of treatment on these primary RPECs are examined on invertedmicroscope using phase contrast optics. They are photographed with T-Max400 film (Kodak) at zero, five, and ten minutes and at twelve hourspost-treatment. The RPECs are not significantly affected by ten minutesexposure to the treatment solutions. FIG. 9 depicts a representativeexample of the effects of the treatment on these cells. Photograph 9Adepicts RPECs at zero minutes. Photograph 9B depicts RPECs at fiveminutes posttreatment. Photograph 9C depicts RPECs ten minutespost-treatment. Photograph 8D depicts RPECs after the cells were washedfollowing treatment.

Example 10

To assess whether there is any side effect of this treatment on otherocular cells, confluent monolayers of LECs, CEDCs and RPECs are preparedas described in Example 1. These cells were exposed for about tenminutes to 200 μl/well of treatment solutions comprising ≧200 mM NaCl,90M frusemide, and 3 μl of 5N NaOH.

The viability of the treated cells was determined by MTT assay 12 to 24hours post-treatment as described in Example 1. Cell survival responseof LECs, RPECs and CEDCs to the treatment are depicted in FIG. 10. Eachpoint represents the mean±s.e.m. of four experiments. As shown, tenminutes after treatment, the treatment solution induced substantiallygreater incidence of cell detachment and/or death, measured by cellviability, of LECs than CEDCs and RPECs. Specifically, more than seventypercent of CEDCs and eighty percent of RPECs survived after thetreatment. This can be compared to a less than ten percent survival ratefor LECs.

Example 11

The effects of treatment on organ cultured corneal buttons is examinedto determine whether the treatment would cause damage to surroundingocular tissues in an environment close to in vivo. Cornea is chosenbecause of its sensitivity and the danger of direct exposure totreatment. Corneal organ cultures are washed with HBSS and placedendothelial-side up on a tailor-made stainless steel stand. They arethen stored in a sterile culture dish containing MEM growth mediumcovering only the bottom of the culture dish, providing a humidifiedchamber to prevent drying of the epithelium. The endothelial cornealconcavity is then filled with MEM growth medium supplemented with 10% to15% FCS, and cultured at 37° C. in a 5% CO₂ incubator.

The organ cultures are divided into control and treatment groups. Thecultures are first washed three times with HBSS and then theirendothelial surfaces are exposed for ten minutes to either physiologicalNaCl (137 mM, 293±9 mOsm/L) or the treatment solution.

For the treatment group, ten milliliters (10 mL) of treatment solutionscomprising (i) ≦200 mM NaCl, (ii) about 90 μM frusemide or about 112 μMbumetanide, and (iii) 3 μl of 5N NaOH are prepared in a deep 35 mmpetri-dish. The organ cultures are transferred to the petri-dishcontaining the treatment solution and are kept in the treatment solutionfor more than ten minutes. The organ cultures are then transferred usingforceps to another petri-dish containing fresh HBSS. The corneal organcultures are then thoroughly rinsed with a fresh HBSS solution withslight pressure using an aspiration/irrigation probe. After thetreatment, the corneal organ culture is incubated in fresh medium forsix hours.

For the control group, instead of the treatment solution describedabove, a fresh HBSS was applied to the corneal organ cultures for tenminutes.

To assess toxicity of the treatment on corneal endothelial cells, thecorneal organ culture buttons are examined by both inverted microscopeusing phase contrast optics and fluorescent-confocal microscopy. Underconfocal microscope, the morphology of actin filaments of cornealendothelial cells is examined to establish evidence for the intactarchitecture of the cells. The endothelium of these corneal organcultures is fixed before and after treatment, and stained withFITC-conjugated phalloidin. Confocal laser-scanning images of cornealendothelium demonstrate no significant differences between untreated(control) and treated groups. The endothelial sheet in the treated groupmaintains the same integrity as those in the untreated group. Thecorneal endothelial cells remain as an evenly distributed monolayer withthe cells keeping their hexagonal appearance in the control as well asin the treated organ cultures. There is no apparent damage to cornealendothelium cells.

Example 12

The in situ effects of a treatment solution comprisingdihydrochlorothiazide (DHCT) (a cotransport interference agent from thegroup of the thiazide and related diuretics) and hyperosmotic NaCl onhuman LECs is examined. The treatment is tested on LECs that are stillattached to a continuous curvilinear capsulorhexis lens capsule(LECs-CCC) which are freshly removed from a human cataract operation.

The LECs-CCCs are obtained from a cataract operation and immediately putin freshly prepared Eagle's Minimum Essential Medium (MEM) supplementedwith 2 mM glutamine, 25 mmol/L Hepes (pH 7.4) without foetal cow serum(FCS) and is transferred to the laboratory for evaluation. Viable LECsare identified by staining the LECs-CCCs for five minutes with 0.2%Trypan Blue. The sheets containing more than 70% viable LECs are used toassess the efficacy of the treatment.

The treatment solutions are composed of either 170 or 200 mM NaCl indH₂O and 200 or 300 μm DHCT.

In the first step, a hyperosmotic solution of NaCl is prepared by makinga stock solution of 2 M NaCl by adding 58.44 g NaCl (supplied by SigmaChemical Co., U.S.) to 500 ml dH₂O. The desired concentration of osmoticNaCl is prepared by diluting the 2M stock solution with dH₂O. Forexample, to make a hyperosmotic solution of 170 mM NaCl, the 2M NaClstock solution was diluted with dH₂O at a ratio of 1 to 10.1. To make ahyperosmotic solution of 200 mM NaCl, the 2M NaCl stock solution isdiluted with dH₂O at a ratio of 1 to 9. Final osmolarities (mOsm/kg/H₂O)are determined by a freezing point depression method using cryoscopicosmometry.

In the second step, DHCT stock solution is made by adding 25 mg DHCT to1 ml dH₂O.

In the final step, the treatment solution with 200 or 300 μm DHCT ismade by diluting the DHCT stock solution in the 170 mM or 200 mMhyperosmotic NaCl and the pH is adjusted to ±7.8.

The treatment is applied by exposing the LECs-CCCs for five minutes in asmall petri dish containing the treatment solution. The treatmentsolution is removed after five minutes exposure by a syringe and thenthe fresh PBS is added in the petri dish to wash away the treatmentsolution. The LECs-CCCs are washed three times. The changes of the LECSare monitored under a microscope. Microscope photographs are taken at 0,2 and 5 minutes and after wash.

After exposure to the treatment solution, the human LECs shrink andgradually large gaps form between the cells. The shrinking cells thengradually round up and burst.

FIG. 11 shows changes of LECs-CCCs caused by a treatment solutioncontaining 200 μm DHCT and hyperosmotic ≦200 mM NaCl at (A) zerominutes, (B) two to three minutes and (C) after wash. In FIG. 11A, whichshows the lens cells before treatment, the lens cells are tightly packedand in close contact with adjacent cells and resemble an intact sheet.The inset in FIG. 11A is a higher magnification of the image. FIG. 11B,taken two to three minutes post-treatment, shows the cells shrinkingwith large gaps between cells. The inset in FIG. 11B is a highermagnification of the image. In FIG. 11C, taken after rinsing away thetreatment solution and the detached cells, no living cells can beobserved aside from some remaining cell debris. The inset in FIG. 11C isa higher magnification of the image.

Example 13

The in vivo efficacy of the treatment described in Example 12 on theprevention of PCO is examined by experiments on two groups of NewZealand albino rabbits. The experimental group includes sixteen rabbiteyes and the control group includes four rabbit eyes. A sham cataractoperation is performed on the control animals. The therapy is introducedto the others.

The pupil of each rabbit eye is preoperatively dilated with topical GPhenylephrine 10% (Alcon Labs., Inc., China) and G Cyclopentolate 1%(Bausch & Lomb Pharm., Inc., USA) three times in one hour. The rabbitsare anesthetized with ketamine hydrochloride (5 mg kg-1) and xylazinehydrochloride (2 mg kg-1) and placed on an operating table. A topicalanesthetic (Dieaine hydrochloride 1%) is used every five minutes on twooccasions. A wire lid speculum is placed to hold the eyelids open.

A 3.2 mm scleral tunnel incision into the eye is formed at the temporallimbus. The anterior chamber is maintained with viscoelastic substancesand a 4-5 mm diameter continuous curvilinear capsularhexis (CCC) is madeon the anterior surface of the lens to open its bag. The treatmentsolution is first introduced in the LECs by hydrodissection. Thisensures that the treatment fluid immediately reaches under the entireanterior capsule/LECs. This process allows for (i) separation of thelens cortex from the capsule and lens epithelium, making removal of thelens cortex easier at a later stage of the operation and (ii) the LECsto be exposed to the treatment for enough time (more than five minutesin total time).

The crystalline lens is then removed using phacoemulsification(Universal II, Alcon Labs., Inc., China). The remaining cortex isaspirated thoroughly and removed as completely as possible to expose theLECs. The lens capsular bag is then partially filed with viscoelasticmaterial in order to maintain its shape. The viscoelastic materialshould not cover the LECs when the treatment solution is applied.

The treatment is spread over the inner surface of the anterior capsularbag (where the LECs attach). The anterior capsular is continuouslyfilled by using a syringe with a bent needle. Care is taken not to overflow the bag. After the treatment solution covered the capsule bag, itis left for five minutes and then removed using a syringe.

In a few cases, IOLs are inserted. In those cases, after the solutionhas covered the whole inner surface of the capsule bag, the treatmentsolution is carefully removed after 3 to 5 minutes by syringe. At thesame time, the incision is enlarged to 6.0 mm and a posterior ocularlens (IOLs) (poly-methyl methacrylate, PMMA) with a 6.5 mm optic isprepared and placed into the capsular bag. Because this procedurenormally takes a surgeon about five minutes, the LECs should have beenexposed to the treatment solution for approximately ten minutes.

The capsular bag is then thoroughly rinsed with slight pressure to clearup the detached and dead LECs and remove excess treatment solution andviscoelastic material. The wound is closed with or without suture.

At the completion of the surgical procedure, each rabbit eye receivessubconjunctival dexamethasone (0.25 ml) and gentamicin (0.25 ml).Postoperatively, atropine sulphate 1% ophthalmic solution and neomycinsulphate-polymyxin sulphate-dexamethasone ophthalmic ointment areadministered twice daily for three weeks.

The animals are evaluated for three weeks and ten weeks. Three weeks issuggested in the literature to be optimal to demonstrate thepreventative effect on PCO in rabbits. All rabbits are evaluated byslitlamp and scored for ocular responses at 1 day and at 1, 2 and 3weeks. (See Table 1.) Three and ten weeks after surgery, the animals areanesthetized using a 2 ml intramuscular injection of 11:1 mixture ofketamine hydrochloride and xylazine 20 mg/ml and then killed with a 2 mlintravenous injection of sodium pentobarbital.

The eye globes are enucleated (some of them fixed in neutral bufferedformalin 10% solution for 24 hours) and are bisected coronally justanterior to the equator. Gross examination is performed to assess. PCOdevelopment. Photographs are taken from behind (Miyake-Apple posteriorphotographic technique) by using a digital camera fitted to an operatingmicroscope (Leica/Wild MZ-8, Vashaw Scientific, Inc.).

The extent and severity of PCO is scored (see Table 1) according tomethods reported in the literature. The lens capsules are then fixed andevaluated under a microscope (Olumpus, Optical Co. Ltd.) andphotographed with a digital camera. The absence or presence of cells,cell types, and the extent of cellular growth are determined.

The group treated with the treatment solution reveal minimal intraocularinflammatory reaction and deposits on the lens capsule surface at both 3and 10 weeks post-treatment. No central PCO(CPCO) (the area within thepupillary area) and peripheral PCO (PPCO) (within 6-6.5 mm outside thepupillary area) is found to have developed three or ten weekspost-treatment. (See FIGS. 12C and 12D). Most eyes did develop somedegree of Soemmering's ring associated with the amount of residual lenscortical fibers.

FIG. 12C is a gross photograph from behind the eye showing a treatedrabbit eye after small incision cataract operation (no IOLs insertion)three weeks after surgery. The pupil area is clear. FIG. 12D is a grossphotograph after removal of the iris. There is an absence of PPCO beyondthe 6 mm central area. FIG. 12E is a phase contrast photomicrograph ofthe pupil area shown in FIG. 12C in which there appears to be noremaining lens epithelial cells.

In contrast to the treatment group, the eyes in the control groupdeveloped dramatic CPCO and PPCO. FIG. 12A is a gross photograph (takenfrom behind the eye using the Miyake-Apple technique) of a controlrabbit eye 3 weeks after cataract operation (no IOLs insertion). Thereis significant fibrous from PCO in the pupil area. FIG. 12B, shows thatunder the microscope, there is significant proliferation of mixedpigmented and lens epithelial cells in the control eye, covering theentire lens capsule. (See FIG. 12B.)

Example 14

The in situ effects of a treatment solution comprising 308 μm bumetanide(a cotransport interference agent from the group of loop diuretics) andhyperosmotic 170 or 200 mM NaCl in dH₂O on LECs-CCCs is examined. TheLECs-CCCs are immediately used after being freshly removed from cataractoperation and treatment is applied as in Example 12.

After exposure to the bumetanide-hyperosmotic NaCl treatment solution,the LECs shrink and gaps form between the cells. The shrunken cells donot round up but fix or arrest in the shrunken state on the lenscapsule. The LECs do not detach, but arrest and die while shrunken. (SeeFIG. 13.)

FIG. 13 shows the cellular changes of the LECs induced by treatment at(A) zero minutes, (B) five minutes and (C) after washing (fifteenminutes). FIG. 13A shows an intact lens epithelium sheet before thetreatment with a few detached LECs, red blood cells and cell debriscaused by the cataract operation. The inset is higher magnification ofthe image. FIG. 13B is taken five minutes post-treatment and shows cellshrinkage and large gaps between the cells. The inset is highermagnification of the image. FIG. 13C is taken fifteen minutes afterwashing away the treatment solution and any detached cells. The imageshows the same appearance of shrinking cells as seen in FIG. 13B. Theinset is higher magnification of the image. The cells died aftertreatment as confirmed by assessment by trypan blue exclusion.

Example 15

The in vivo efficacy of the treatment solution described in Example 14(bumetanide and hyperosmotic NaCl) on the prevention of PCO is examinedby experiments on two groups of New Zealand albino rabbits. Thetreatment group includes five rabbit eyes and the control group includestwo eyes, established and assessed in parallel. The same surgical andevaluative procedures described in Example 13 are used in thisexperiment.

In three weeks follow up, the experiments reveal that no CPCO and PPCOdevelop in treated rabbits (observations made by gross examination frombehind the eye using Miyake-Apple technique). (See FIG. 14.) Consistentwith the results in situ (see Example 14), after exposure to thebumetanide-hyperosmotic NaCl solution, the whole monolayer of the lensepithelium is arrested in a shrunken appearance. This is only observedin the lens anterior capsule. (See FIG. 14B.) Not many cells in the lensposterior capsules are observed. Also, there is not significant cellproliferation on the lens anterior capsule although there is a monolayerof LECs with the described shrunken appearance.

FIG. 14A, a gross photograph taken from behind the eye using theMiyake-Apple technique, shows a clear pupil are in the treated rabbiteye three weeks after the cataract operation (no IOLs insertion). FIG.14B, a phase-contrast photomicrograph taken from such pupils of the lenscapsules after treatment, shows two layers of capsules. The top layer isthe anterior capsule with a layer of shrunken cells. The bottom layer isthe posterior capsule where there are no clear cells observed. Theinsets are higher magnifications of the images. The inset for FIG. 14Bshows a monolayer of cells with a shrunken appearance and no significantproliferated cells.

Some degree of Soemmering's ring forms in the treated eyes, whichappears associated with an ineffective cortical clean-up procedure.

In contrast to the results observed in the treatment eyes, there issignificant fibrous growth from PCO observed in the pupil area of thecontrol eyes, which are evaluated three weeks after cataract operation(no IOLs insertion).

Example 16

The in situ effects of a treatment solution comprising 200 or 300 μmN-ethylmaleimide (NEM) (a cotransport interference agent that activatesor stimulates the efflux of KCl) in PBS on LECs-CCCs is examined.Treatment is applied as in Example 12.

After five minutes of acute exposure to the NEM treatment solution,almost no changes are observed in the LECs. However, once the treatedLECs are put into a PBS washing solution, they shrink and large gapsform between the cells. The shrunken cells then detach from the LECscapsule as found in those described in Example 12.

FIG. 15 shows the cellular changes of the LECs-CCCs induced by the NEMtreatment at (A) zero minutes, (B) two to three minutes and (C) twominutes after wash. FIG. 15A shows an intact lens epithelium sheetbefore treatment with some damages and detached LECs and cellular debriscaused by the cataract operation. FIG. 15B, taken two to three minutespost-treatment, shows slight cell shrinkage. Once the cells are placedback in fresh PBS washing solution, the cells shrink significantly andlarge gaps form between the cells. These cells then detach and wash awayeasily at the end of the experiments.

Example 17

The in vivo efficacy of the treatment solution generally described inExample 16 (NEM in PBS) on the prevention of PCO is examined byexperiments on two groups of New Zealand albino rabbits. The treatmentgroup includes six rabbit eyes and the control group includes two rabbiteyes, established and assessed in parallel. The surgical and evaluativeprocedures in Example 13 are used.

The eyes in the treatment group are treated with 200 or 300 μm NEM inPBS or 170 mM monnitol. In 3 and 10 weeks follow up, no CPCO or PPCOdevelops in the treated rabbits (observed in gross examination frombehind the eyes). (See Table 1 and FIG. 15D.) The pupil area of treatedeyes is clear. There are no LECs on the lens capsule bag when it isexamined under microscope. (FIG. 15E.) However, the treated eyes exhibita heavy intraocular inflammatory reaction in the early period andposterior synechiae.

In contrast to the results observed in the treatment eyes, there issignificant fibrous growth from PCO observed in the pupil area of thetwo control rabbit eyes (no IOLs insertion).

Example 18

The procedure by which treatment is applied to in vivo eyes is modifiedto prevent heavy anterior chamber (AC) reaction. Instead of introducingthe treatment solution directly into the eyes, the IOLs are coated withtreatment solution and are inserted into the lens capsular bag after thelens contents are removed. Specifically, the posterior and equatorportion of the IOLs are coated with the treatment solution containing300 μm NEM in PBS. They are then dried overnight in a sterile Laminarflow hood. The IOLs are then ready to use. This procedure significantlyreduces the AC reaction.

Example 19

The in situ effects of a treatment solution comprising 10 or 100 μmgramicidin (an agent from the class of pore-forming proteins andpeptides (PFPs)) and PBS on human LECs is examined. Treatment is appliedas in Example 12.

The LECs-CCCs are exposed to the gramicidin-PBS treatment solution forfive minutes. Changes to the LECs are monitored under microscope. FIG.16 shows microscope photographs taken at (A) zero minutes (B) twominutes and (C) after wash.

After exposure to the treatment, the human LECs shrink and large gapsgradually form between the cells. The shrinking cells then graduallyround up and burst. FIG. 16A, taken before treatment solution isapplied, shows lens cells in tight contact with each other andresembling an intact sheet of cells. FIG. 16B shows that, at two minutesafter exposure to the treatment solution, the cells take on a shrunkenappearance and clear gaps between the cells form. FIG. 16C shows that,after the treatment solution and detached cells have been rinsed away,no living cells remain apart from some remaining cell debris.

Many modifications and variations of the present invention are possiblein light of the above teachings. For example, other ion transportmechanism interference agents may be used than those discussed above indetail such as the following which may be appropriate: valinomycin,bromine, theophylline and other alkylxanthines, icosanoids, phorbolester (e.g. TPA), potassium-sparing diuretics (e.g., triamterene,spironolactone, and potassium canrenoate), calyculin A, okadaic Acid(OA), propranolol and its analogs, Angiotensin II, Ketoconazole (CDC),arachidonic acid, ianthanum, trifluoperazine, idoxifene,2-aminisobutyric acid, 17 beta-oestradiol, Bradykinin,phosphatidylinositol 4,5-biphosphate (PIP2), inositol 1, 4, 5,triphosphate (IP3), prostaglandins (PGE₂), adenosine 3‘5’-cyclicmonophosphate (cAMP), guanosine 3′5′-cyclic monophosphate (cGMP),serine/threonine protein phosphatases (S/T-Ppases), protein kinase C(PKC), protein kinase A (PKA), mitogen-activated protein kinase (MAPK),SRC family tyrosine kinases (SFKs), polyunsaturated fatty acids,phospholipase C (PLC), phosphatases (PP), G-proteins, Rubidium, Copper(Cu²⁺), Nitrate (NO³⁻), Barium (Ba²⁺), Chloride (Cl⁻), Potassium (K⁺),agents that change intracellular magnesium and hydrogen concentration,and agents that decrease extracellular sodium, chloride or potassiumconcentrations. It should be appreciated that there are other iontransport mechanism interference agents as well. Thus, it is to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as described hereinabove.

1. An ocular treatment solution, having a pH of about 7 to 10,comprising: an ion transport mechanism interference agent and anosmolyte wherein the solution induces substantially greater incidence ofdetachment of lens epithelial cells than corneal endothelial cells andretinal pigmented epithelial cells to prevent posterior capsularopacification and wherein the lens epithelial cells shrink prior todetachment.
 2. An ocular treatment solution as recited in claim 1wherein the ion transport mechanism interference agent is a co-transportinterference agent.
 3. An ocular treatment solution as recited in claim2 wherein the co-transport interference agent is selected from the groupof diuretics.
 4. An ocular treatment solution as recited in claim 3wherein the co-transport interference agent is dihydrochlorothiazide. 5.An ocular treatment solution as recited in claim 4 wherein the osmolytecomprises an inorganic salt.
 6. An ocular treatment solution as recitedin claim 5 wherein the treatment solution has a hyperosmotic osmolarityand wherein dihydrochlorothiazde is present in the solution in aconcentration ranging from about 60 μM to about 500 μM.
 7. An oculartreatment solution as recited in claim 3 wherein the co-transportinterference agent is bumetanide.
 8. An ocular treatment solution asrecited in claim 8 wherein the osmolyte comprises an inorganic salt. 9.An ocular treatment solution as recited in claim 8 wherein the treatmentsolution has a hyperosmotic osmolarity and wherein bumetanide is presentin the solution in a concentration ranging from about 30 μM to about 500μM.
 10. An ocular treatment solution as recited in claim 2 wherein theco-transport interference agent is n-ethylmaleimide.
 11. An oculartreatment solution as recited in claim 10 wherein n-ethylmaleimide ispresent in the solution in a concentration ranging from about 100 μM toabout 500 μM.
 12. An ocular treatment solution as recited in claim 1wherein the ion transport mechanism interference agent comprises apore-forming protein or peptide.
 13. An ocular treatment solution asrecited in claim 12 wherein the pore-forming protein or peptide isgramicidin.
 14. An ocular treatment solution as recited in claim 13wherein gramicidin is present in the solution in a concentration rangingfrom about 10 μM to about 300 μM.
 15. A method of using an oculartreatment solution as recited in claim 1 wherein the solution is appliedto an intraocular lens prior to surgery.
 16. An ocular treatmentsolution comprising: an ion transport mechanism interference agent andan osmotic stress agent to prevent posterior capsular opacification, thesolution having a pH from about 7 to about 10 and the solution rapidlyinducing death via necrosis of lens epithelial cells.
 17. A method forpreventing posterior capsular opacification comprising: applying atreatment solution comprising an ion transport mechanism interferenceagent and an osmotic stress agent to an intraocular lens; and placingsaid intraocular lens into the eye wherein there is substantiallygreater incidence of detachment of lens epithelial cells than cornealendothelial cells and retinal pigmented epithelial cells to preventposterior capsular opacification.
 18. An ocular treatment solution asrecited in claim 1 wherein the ion transport interference agent ispresent in the solution in a concentration less than about 1500 μM. 19.An ocular treatment solution as recited in claim 1 wherein the iontransport interference agent is present in the solution in aconcentration less than about 1000 μM.
 20. An ocular treatment solutionas recited in claim 1 wherein the ion transport interference agent ispresent in the solution in a concentration less than about 500 μM. 21.An ocular treatment solution as recited in claim 3 wherein the diureticis a thiazide diuretic.